A need exists to identify novel lead compounds that are capable of modulating pharmaceutical and agricultural molecular reactions. Historically, such novel compounds were identified by individually synthesizing a compound and testing it for biological activity. This time-consuming process has been replaced, in part, by a process referred to as "rational drug design". This traditional route to drug discovery requires a knowledge of the structure of the target (i.e., ligate) and/or its cognate (i.e., ligand) in order to synthesize only those molecules which are structurally-related to known ligands of the biological target. However, despite this more focussed approach to identifying lead compounds, the time requirements for conventional structural analysis and chemical synthesis of complex molecules continues to contribute to the substantial periods of time consumed by research and development in the discovery of novel drugs.
The application of recombinant technology and automated methods for solid phase chemical synthesis to the generation of molecularly diverse libraries has circumvented many of the most time-consuming steps associated with the traditional drug discovery process. As a result of these technological advances, "rational drug design" rapidly is being replaced by "irrational drug design", i.e., the process of screening libraries of molecularly diverse molecules to identify biologically active molecules contained therein (Brenner, S., and Lerner, R., Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992)).
Recombinant molecular libraries are generated by inserting random segments of nucleic acid into a vector, such as a phage, and allowing the vector to replicate, transcribe and express the inserted sequence. In a phage library, the nucleic acid is inserted into the phage genome in a manner which permits expression of the inserted material on the phage surface (see, e.g., Pavia, M., et al., Bioorg. & Med. Chem. Ltrs. 3(3):387-396 (1993) and references cited therein; Devlin, J., et al., Science 249:404-406 (1990)). Typically, the virus particles are screened for the presence of a lead compound by contacting the particles with an immobilized ligate and isolating the virus particles which express a ligand that binds to the immobilized ligate.
The primary advantage of using a recombinantly-produced library for drug discovery is the ability to clone and amplify the nucleic acids encoding the lead compounds which are identified during the screening process. Unfortunately, recombinant libraries inherently are limited in diversity to linear polymers (e.g., oligonucleotides, peptides) formed of naturally-occurring monomers (e.g., nucleotides, L-amino acids). Typically, these naturally-occurring polymers exhibit metabolic instability and poor absorption properties in vivo. Accordingly, pharmaceutical lead compounds that are isolated from recombinant libraries frequently require substantial modification to obtain a clinically useful drug.
To overcome these limitations, a number of chemical methods have been used to generate molecular libraries. In general, such methods utilize a solid support (e.g., cellulose paper, cotton, polystyrene-grafted polyethylene film, polymeric beads and resins) upon which a diverse collection of linear compounds are synthesized using conventional coupling chemistries (e.g., Merrifield, J. Am. Chem. Soc. (1963) 85:2149-2154). The synthesis of libraries containing molecularly diverse peptides has become commonplace since Geysen et al. first reported the chemical synthesis of a peptide library on polyacrylic acid grafted polyethylene pins (Geysen, H., et al., J. Immunolog. Meth. 102:259-274 (1987)). More recently, Houghten et al. (BioTechniques 4(6):522-528 (1986) and Nature 354:84-86 (1991)) described a method for generating peptide libraries in which small amounts of a resin support were encapsulated in a plurality of porous polypropylene bags. By sequentially immersing the bags in solutions of individual amino acids under conditions for forming a peptide linkage, Houghten et al. generated a collection of bags, each of which contained a unique resin-immobilized peptide. The generation of combinatorial bead-immobilized peptide libraries also has been reported using a split synthesis procedure (Lam, K., et al., Nature 354:82-84 (1991). Solid-phase peptide synthesis also has been combined with photolithography to prepare arrays of peptides or oligonucleotides attached to solid supports (Fodor, S., et al., Science 251:767-773 (1991)).
Although chemically-synthesized libraries offer nearly unlimited molecular diversity, it typically is necessary that small library molecules be cleaved from the solid support prior to assessing biological activity. Moreover, the above-recited methods typically produce libraries of linear polymers in which monomers are connected via a biological backbone (e.g., a polypeptide backbone of amide linkages connecting amino acid monomers). Because such structures are recognized by degradative enzymes in vivo, many chemically-synthesized library molecules also are metabolically unstable.
Frequent attempts have been made to identify compounds which inhibit the enzymatic activity of proteases and in particular, to identify inhibitors of serine proteases. The serine proteases are a family of enzymes that utilize an activated serine residue in their substrate binding domain to hydrolyze peptide bonds. See, e.g., "Textbook of Biochemistry", 3rd edition, ed. T. Devlin, N.Y., N.Y., John Wiley & Sons, Inc., pp.102-116 (1992). The Devlin textbook, as well as each of the references, patent publications and patents identified in this application, are incorporated in their entirety herein by reference.
Abnormally high levels of serine proteases have been implicated in the following physiological conditions (the serine protease believed to mediate the condition appears in parentheses): pancreatitis (trypsin, chymotrypsin, pancreatic elastase, enterokinase); cerebral infarction, coronary infarction, thrombosis, bleeding disorders (plasma kallikrein, Factors XIIa, XIa, IXa, VIIa, Xa and IIa (thrombin) and Activated Protein C); inflammation, rheumatoid arthritis, autoimmune disease (Factors C1r, C1s, D, B and C3 Convertase); clotting disorders, tumor metastasis (urokinase plasminogen activator, tissue plasminogen activator, plasmin); infertility (acrosin); and inflammation, allergic response (granulocyte elastase, Cathepsin G, mast cell chymases, mast cell tryptases). Accordingly, agents which reduce or eliminate the catalytic activity of these enzymes, i.e., serine protease inhibitors, are useful for treating individuals diagnosed as having the above-mentioned serine protease-mediated physiological conditions.
Several naturally-occurring serine protease inhibitors have been identified and isolated. These inhibitors are believed to play a key role in the regulation of serine protease activity in vivo. In view of the clinical significance of the serine protease inhibitors, early efforts to identify serine protease inhibitors focussed on isolating peptide inhibitors from mammalian tissue, chemically synthesizing inhibitors having the same amino acid sequence as the natural peptide inhibitors and/or chemically (or otherwise) modifying the natural (or chemically equivalent) peptide inhibitors.
U.S. Pat. No. 4,963,654, issued to Katunuma, describes a naturally-occurring trypsin inhibitor that is isolated from animal tissue. The inhibitor is a basic peptide containing L-amino acids and has a molecular weight of about 7,600. U.S. Pat. No. 5,157,019, issued to Glover et al. also describes novel peptides which exhibit inhibitory activity toward serine proteases. The Glover et al. inhibitory peptides include a generic inhibitory amino acid core sequence that is recognized by several different proteases. The inhibitory core sequence contains between about eleven and about thirty-one amino acid residues. U.S. Pat. No. 5,240,913, issued to Marganore et al. describes a thrombin inhibitor which contains naturally-occurring, as well as non-naturally occurring amino acids.
Chemically synthesized serine protease inhibitors are disclosed in U.S. Pat. No. 5,250,677, issued to Han (hereinafter "Han '677") and U.S. Pat. No. 5,109,018, issued to Powers et al. (hereinafter "Powers et al. '018"). Han '677 describes the synthesis of azetidin-2-one derivatives and their use as serine protease inhibitors. In particular, Han '677 report that the derivatives exhibit anti-thrombin and anti-trypsin activities and are useful in controlling blood coagulation and treating pancreatitis.
Powers et al. '018 describes the synthesis of heterocyclic compounds and reports that these compounds inhibit serine proteases having chymotrypsin-like, elastase-like and trypsin-like specificities. Powers et al. further report that these compounds are useful for treating physiological conditions which involve tissue proteolysis (e.g., emphysema), as well as for preventing undesired proteolysis that occurs during the production, isolation, purification, transport and storage of valuable peptides and proteins.
A biotransformed heterocyclic compound, described in U.S. Pat. No. 5,192,668, issued to Treiber et al., reportedly exhibits inhibitory activity toward a protease present in the HIV virus. The biotransformed molecule is prepared by culturing a particular Streptomyces culture in the presence of a known HIV protease inhibitor to yield a biotransformed molecule having anti-HIV protease inhibitor activity.
Despite the developments in genetic engineering, rational drug design and the recognition of the clinical and industrial value of developing more potent, biologically-stable serine proteases, few serine protease inhibitors having the requisite activity for inhibiting the catalytic activity of proteases in vivo and/or in vitro to accomplish the above-described clinical and industrial objectives have been identified. Accordingly, a need exists to identify, select and manufacture serine proteases inexpensively and in commercially reasonable quantities to achieve these goals.